Cilia Assembly and Transport in the Vertebrate Retina

描述(申请人提供)：这个项目的长期目标是了解脊椎动物感光细胞中纤毛形成和维持的分子基础。在脊椎动物中，光感受器外节的组装和维持始于连接纤毛的形成。连接纤毛含有微管为基础的轴丝，该轴丝由一个基准体固定在顶端内段。纤毛的形成始于基体在内节顶面的对接。基因突变破坏了基本体和/或纤毛的组装、结构或功能，导致了一系列称为纤毛病的疾病。这些多综合征疾病通常表现为视网膜变性、肾脏疾病、智力低下和多指畸形。在目前的应用中，我们将利用斑马鱼的功能丧失策略来研究控制基底体定位的机制。在特定的目标1中，我们将通过研究斑马鱼Dynein和dynactin的p150和p50亚基的突变来检验这一假设，即dynein/dynactin复合体调节纤毛形成之前基底体的顶端运输。在特定目标2中，我们提供了在成年斑马鱼视网膜内显示高度极化排列的基本小体的初步证据。我们将直接测试这一假设，即PCP途径调节这种模式，并对光感受器生存至关重要。在特定的目标3中，我们将检查携带Joubert综合征基因Arl13b零突变的斑马鱼的视网膜表型。拟议的实验还将测试GTPase结构域和纤毛靶向序列RVxPx对Arl13b功能的要求。我们还将测试Arl13b与Bardet-Biedl综合征(BBS)基因和PCP途径组件的功能相互作用。这些相互作用将确定潜在的第二位点修饰，以增强光感受器表型的表达。这些研究的结果将揭示纤毛形成前基础体位所需的新机制，并确定影响遗传性失明的新的遗传交互作用。 与公共健康相关：基本身体的对接和锚定对于脊椎动物光感受器连接纤毛和外节的形成是必不可少的。纤毛/基底体结构是一个复杂的细胞器，具有重要的临床意义，因为纤毛的位置、装配或功能缺陷会导致视网膜变性、肾脏疾病、智力低下、内翻、多指等情况。了解纤毛形成的控制机制将有助于开发治疗睫状体疾病的方法。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the molecular basis of cilia formation and maintenance in vertebrate photoreceptor cells. In vertebrates, the assembly and maintenance of photoreceptor outer segments begins with the formation of a connecting cilium. The connecting cilium contains a microtubule- based axoneme that is anchored to the apical inner segment by a basal body. Cilia formation begins with the docking of basal bodies at the apical surface of the inner segment. Genetic mutations disrupting the assembly, structure, or function of basal bodies and/or cilia result in a spectrum of diseases known as ciliopathies. These multisyndromic disorders often present with retinal degeneration, kidney disease, mental retardation, and polydactyly. In the current application, we will utilize loss-of-function strategies in zebrafish to investigate the mechanisms controlling basal body localization. In Specific Aim 1, we will test the hypothesis that the dynein/dynactin complex regulates the apical transport of basal bodies preceding cilia formation by examining zebrafish mutants in dynein and the p150 and p50 subunits of dynactin. In Specific Aim 2, we provide preliminary evidence that basal bodies show a highly polarized arrangement within the adult zebrafish retina. We will directly test the hypothesis that the PCP pathway regulates this patterning and is essential for photoreceptor survival. In Specific Aim 3, we will examine zebrafish carrying null mutations in the Joubert Syndrome gene arl13b for retinal phenotypes. Proposed experiments will also test the requirement of the GTPase domain and a ciliary-targeting sequence RVxPx for Arl13b function. We will also test arl13b for functional interactions with Bardet-Biedl Syndrome (BBS) genes, and components of the PCP pathway. These interactions will identify potential second-site modifiers that enhance expression of photoreceptor phenotypes. The results of these studies will reveal novel mechanisms required for basal body placement prior to cilia formation and to identify novel genetic interactions that influence hereditary blindness. PUBLIC HEALTH RELEVANCE: The docking and anchoring of the basal body is essential for the formation of the connecting cilium and outer segment of vertebrate photoreceptors. The cilia/basal body structure is a complex organelle of great clinical importance because defects in cilia positioning, assembly or function lead to retinal degeneration, kidney disorders, mental retardation, situs inversus, polydactyly, and other conditions. An understanding of the mechanisms that control the cilia formation will lead to the development of treatments for ciliary diseases.